Summary
OBJECTIVEApoptosis is considered the main factor inducing by bortezomib in myeloma cells. Although bortezomib (BTZ) is a crucial drug for the treatment of MM, chemoresistance is a major problem. OncomiR- 19a plays an oncogenic role in many types of cancer such as MM; however, the function of miR-19a in the pathogenesis of MM and drug resistance has not been identified well. The present research aims at investigating the inhibition of miR-19a by antagomir to conclude BTZ responsiveness and suggests that miR-19a may be a biomarker for the prognosis of the patient.
METHODS
In the present research, the viability and apoptosis of myeloma cells were analyzed by colorimetric MTT
and Annexin-PI flow cytometric assays. QRT-PCR was implemented to evaluate the expression level of
miR-19a, its targets SOCS3 and STAT3, at the mRNA level.
RESULTS
Following treating transfected cells with bortezomib we found out that miR-19a was downregulated,
and the rate of apoptosis of myeloma cells after bortezomib treatment considerably increased. It indicated
that mRNA of SOCS3 increased and STAT3 decreased.
CONCLUSION
The research results indicate that oncomiR-19a as a biomarker may induce better responsiveness to
bortezomib in myeloma cell lines through its targets SOCS3, STAT3, and may provide new therapeutic
targets in the future for myeloma. The research results indicate that oncomiR-19a as a biomarker may
induce better responsiveness to bortezomib in myeloma cell lines through its targets SOCS3, STAT3 and
may provide new therapeutic targets in the future for myeloma.
Introduction
Multiple Myeloma (MM) is some sort of tumor of differentiated B cells from the germinal center, plasma cells, within 10% of all hematologic neoplasms, and is considered the second most commonly occurring non-Hodgkin lymphoma.[1,2] Over the last two decades, due to advancements in available treatments, the median survival time of patients with MM has increased from 3 to 6 years and is accounting for 1.1% of total cancer deaths and the age-standardized risk Of MM is about 2%.[3] Although great discoveries have been made in the growing evolution of new healthcare strategies in the past decade, to a great extent the tumor is still incurable and new therapies are required to overcome it.[4,5] Despite the innovation and benefitting from a new therapeutic strategy such as proteasome inhibitors, the clinical outcome of the patients aggravates, and in most patients finally, MM relapses and engenders drug resistance.[1]The proteasome inhibitor, bortezomib (BTZ), is a crucial and an FDA-approved drug for the treatment of MM, especially relapsed & refractory MM.[6] Although BTZ has a significant impact on the treatment of MM,[5] drug resistance or relapse are still two major challenges, and the prognosis of patients with BTZ resistance is highly undesirable.[7,8] Therefore, to prevent BTZ resistance, new therapeutic methods are desperately required. On the other hand, a more profound molecular grasp of cancer"s pathogenesis is, thus, urgently required to recognize new molecular targets and to bring forth therapeutic agents that are suitable for the patient.[9] In this regard, there is developing evidence indicating that MM stems from the deregulation of noncoding RNAs, including miRNAs.[1] Recent studies show that MM is caused by interruptions in many different signaling pathways, which are driven by the microRNAs and are a class of non-coding RNAs (ncRNAs) of about 18?22 nucleotides in length. These microRNAs act as master regulators of gene expression at the post-transcriptional level via RNA interference pathways.[6] miRNAs are involved in many biological processes, including differentiation, senescence, survival, and, apoptosis.[3,6] It is well established that the disturbance in the regulation of miRNAs is accompanied by the pathogenesis of diseases such as cancer, and miRNA expression profiles have prognostic implications in numerous types of cancer.[6] Altogether, miRNAs play a fundamental role as an oncogene. In other words, miRNAs operate as "oncomiRs" if their targets are the tumor suppressor genes. [1] Therefore, controlling oncomiRs may be an effective strategy for treatment.
Chief among oncomiRs, miR-17-92 clusters that are placed in chromosome 13q31.3, including miR-19a are the very first oncomiRs that were discovered. It is proved that any disturbance in the expression level of miR-17-92 clusters results in the malignant progress of MM.[4,5] MiR-19a, a crucial component of the miR- 17-92 cluster, is directly involved in myeloma pathogenesis and progression of multiple myeloma.[9] Also, besides target genes of miR-19a are considered as potential biomarkers of the disease.[10] Moreover, compared with normal plasma cells, miR-19a has been revealed to be up-regulated in patients with MM, as well as in MM cell lines.
Furthermore, miR-19a can adjust the expression of proteins that are essential for myeloma pathogenesis including suppressors of cytokine signaling (SOCSs).[9] It is reported that miR-19a targets SOCS3, a potent regulator of the JAK-STAT pathway, as is followed by a considerable reduction of SOCS3 mRNA together with enhanced activation of SOCS3 target, STAT3.[9] Based on these findings, a strategy that can be developed to regulate miRNA"s aberrant expression in cancer is the inhibition of up-regulated miRNAs.[1] Therefore, we have assumed that the use of mir-19a inhibitors (Antago-miR) could be a new approach for the treatment of multiple myeloma. On the other hand, based on the use of BTZ in the treatment of patients with MM and the resistance in 60% of them, the present study investigates the impact of antagomiR-19a on better responsiveness to BTZ and may present miR-19a as an effective biomarker for response to treatment.[9,11]
Methods
Cell Lines & Cell CultureIn the present research, the RPMI8226 and U266 cell lines were purchased from the Pasteur Institute of Iran (IPI). The cells were grown in suspension in RPMI1640 medium (BIO-IDEA from Bio Idea Group) supplemented with 10% fetal bovine serum (FBS) (GIBCO- BRL), penicillin (100 mg/mL), streptomycin (100 mg/mL), and 2mM l-glutamine (BIO-IDEA from Bio Idea Group). Cells were maintained at 37°C in an environment of 5% carbon dioxide and 95% air and underwent passage twice a week.
Reagents
Bortezomib (PS-341) (Selleckchem.com Cat.
No.S1013) was dissolved in 0.2603 mL DMSO to prepare
a stock solution with a concentration of 50 mM
for storage at -20°C. LentimiRa-off-has-miR-19a-3p
vector (Applied Biological Materials (ABM) Inc. with
Cat. No. mh30299) with GFP promoter, miRNA insert,
and kanamycin resistance gene that was transformed
in DH5α E.coli strain and isolatedby Qiagen plus Midi
plasmid purification kit and stored at -20°C.
In Vitro Cell Culture and Drug Treatment
Human myeloma cell lines RPMI8226 and U266 were
cultured and 50 mM Stock solution of Bortezomib was
further diluted to working concentrations in RPMI1640
medium before use. To determine the concentration of
drug that inhibited cell proliferation by 50% (IC50), 5×10³ cells were seeded in 96well plate, then treated
with different concentration of working solutions of
BTZ based on the approximate concentration that was
noted in cell assay part of BTZ data-sheet in ranges of
0.5, 5, 50 ?M for U266 and 150, 450, 750, 1050 nM for
RPMI8226, mixed well in RPMI1640 media and 10%
FBS and finally incubated for 48 hours.
MTT Colorimetric Assay
Using a standard protocol, the inhibitory impact of
BTZ on cell growth was assessed by 3-(4, 5-Dimethylthiazol-
2-yl)-2, 5-Diphenyltetrazolium Bromide
(MTT) assay. Briefly, cells from 48-hour cultures were
pulsed with 10 ?L of 5 mg/mL MTT to each well for at
least 4 hours of 48-hour cultures, followed by 100 ?L
of isopropanol containing 0.04 N HCl, absorbance was
measured at 570 nm using a spectrophotometer and results
were expressed as the mean of three replicates as
a percentage of control (taken as 100%). The extent of
cytotoxicity was defined as the relative reduction of the
optical density (OD), which correlated to the number
of viable cells compare to cell control (100%). To decide
the optimum dosage of the drugs for further studies,
the cell viability was plotted in a graph and the IC
50 was calculated accordingly.
Cell Viability Analysis
The effect of transfection on cellular viability was
assessed by flow cytometry using propidium iodide
(PI). PI can only pass through disordered areas of the
dead membrane cells and intercalated with the DNA
of the nuclei, emitting red fluorescence light. It was
used with 1mg/mL concentration by dissolving PI
(Sigma, P 4170) in dH2O. PI solution was added in a
final concentration of 2µg/mL to 1×106 cells in suspension,
incubated at a dark place and after 5 minutes,
analyzed by flow cytometry using Attune NXT
flow cytometer.
Analysis of Apoptosis
The apoptosis of cell lines after transfection with Plenti-
III-miR-Off -has-miR-19a -3p vector, after treatment
with bortezomib and after treatment of transfected
cells with bortezomib compared with untreated cells
were analyzed with Annexin V/PI (eBioscience Cat.
No. 88-8005 from Thermo Fisher Scientific) by flow
cytometry using Attune NXT flow cytometer. Cells
were washed in PBS and then they were washed in 1X
bindings buffer once, after that we resuspended cells
in 1X bindings buffer at 1×106/mL, then we added 5
µL of FITC-conjugated Annexin V to 100 µL of the cell suspension, incubated for 10-15 minutes in a dark
place at room temperature, then they were washed with
binding buffer, resuspended in it afterward we added
5 µL of Propidium Iodide Staining Solution (Sigma, P
4170), analyzed by flow cytometry.
Prediction of SOCS3 as a Target of miR-19a
Target Scan (Version 5) and pic tar were used to confirm
the SOCS3 molecule as a target of miR-19a in its
3´ UTR. Target Scan predicted biological targets of
miRNA by searching for the presence of conserved 8
and 7 base sites that match its seed region.
Cell Transfection
Cells were grown in RPMI1640 medium containing no
antibiotics before transfection. U266 and RPMI8226
were transfected by final concentration 2?g of Plenti-
III-miR-Off-has-miR-19a -3p vector containing
GFP purchased from Applied Biological Materials
Inc. (ABM). Transfection of cells was performed using
UltraCruz® Transfection Reagent (SCBT, Santa
Cruz Biotechnology, Inc.). Briefly, before transfection,
we prepared a transfection reagent and vector
in Opti-MEM I reduced serum medium (Gibco) following
the manufacturer's protocol. After preparing
the transfection reagent and plasmid in OPTI-MEM
I medium and incubation at room temperature, plasmid
reagent was added to transfection reagent, vortex
vigorously incubates for 20 minutes. 6×10³ cells were
added to the Eppendorf tube and were poured above
mix dropwise to the cells, incubated in the incubator
for 2 hours, every half an hour flicked with a fingertip,
finally were transferred to 6-well plate and after 24-72
hours GFP expression was evaluated by Attune NXT
flow cytometer.
Q-RT-PCR Assessment of miR-19a Expression
Total RNA was isolated from un-transfected, transfected
and BTZ treated cells: RPMI8226 and U266 while
following the Trizol manufacturer's protocol (InvitrogenTM,
USA). 2000ng of RNA was reverse transcribed
using specific microRNA stem-loop primers from Qiagen
for miR-19a, to generate cDNA using Hyperscript
reverse transcriptase first-strand synthesis kit (Gene-
All Biotechnology Co., Ltd, Korea). The expression of
Snord47 was used as the internal control for miRNA
expression normalization. The quantitative real-time
PCR was performed with the SYBR® Premix Ex Taq?
miRNA RT-qPCR Detection Kit (Takara, Cat.No.
RR820Q) is using the Qiagen Rotor-Gene Q 5PLEX
HRM Real-Time PCR and primers with sequences matching with table 1. PCR program cycling parameters
included 95° for 15s, 58° for the 30s, 72° for 30s for
45 cycles. Data analysis was done by 2-ΔΔCT to calculate
the fold change of miR-19a relative expression
compared to untreated control.
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Table 1: Primer sequences, product length, and annealing temperature used in the qRT-PCR analysis
Quantitative RT-PCR Analysis for SOCS3, STAT3
Genes
Total RNA was isolated from un-transfected, transfected
and BTZ treated cells: RPMI8226 and U266 following
the Trizol manufacturer's protocol (InvitrogenTM,
USA). 2µg of total RNA was reverse transcribed into
cDNA using Hyperscript reverse transcriptase firststrand
synthesis kit with oligo-dT primers following
the manufacturer's instructions (GeneAll Biotechnology
Co., Ltd, Korea) for evaluation of SOCS3, STAT3
genes. The expression of β-Actin was used as an internal
control. The quantitative real-time PCR was performed
with the SYBR® Premix Ex Taq™ miRNA RT-qPCR Detection
Kit (Takara, Cat. No. RR820Q) using the Qiagen
Rotor-Gene Q 5PLEX HRM Real-Time PCR. PCR
program cycling parameters included 95° for 15s, 58°
for the 30s, 72° for 30s for 45 cycles. Data analysis was
performed by 2-ΔΔCT to calculate the fold change of
the above genes" relative expression compared to untreated
control.
Statistical Analysis
Data were presented as mean±standard deviation. The
statistical analysis was performed with the GraphPad Prism 8.4.0 software The mean values of two groups or
multiple groups were compared by one-way analysis of
variance (ANOVA). P<0.05 was considered statistically
significant. Flow cytometric assays were analyzed with
FlowJo version 7.6.1.
Results
IC50 of BTZ in (U266 and RPMI 8226) MM Cell Lines was 5 ?M and 150 nM, RespectivelyWe treated RPMI 8226 and U266 cell lines with different concentrations of BTZ to optimize its IC50 for the treatment of cell lines with proper concentrations throughout the analysis. By using the MTT assay and viability test with PI through flow cytometry, we found out that the optimized concentration of BTZ for the U266 cell line was 5 µM and 150 nM for RPMI8226 (Fig. 1a, c, and b, d).
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Downregulation of miR-19a Decreased Cell Viability
and Prevented the Proliferation of Myeloma Cell
Lines
We first evaluated the expression level of miR-19a in
non-transfected (control) myeloma cell lines (RPMI
8226 and U266). After the transfection of myeloma cell
lines with the LentimiRa-off-has-mir-19a-3p vector,
the efficiency of transfection was monitored by GFP
evaluation using both fluorescent microscopy (Fig.
2a and 3a) and flow cytometry (Fig. 2b and 3b). The
expression level of miR-19a was then determined in transfected myeloma cell lines (RPMI 8226 and U266)
by qRT-PCR. As shown in fig. 2C and 3C, compared
with the un-transfected myeloma cell line, the expression
level of miR-19a significantly decreased in the
transfected group. The data showed that antago-miR-
19a downregulated expression of miR-19a which is a
previously-known oncomiR. After the transfection of
myeloma cell lines (RPMI 8226, U266) with LentimiRa-
off-has-mir-19a-3p vector, cell viability was evaluated
in 72 hours with PI (Propidium Iodide) and the
rate of viability was detected by flow cytometry (Fig.
2d and 3d). The data confirms the possibility of the
analyses of transfected cell lines within 72 hours after
transfection, while indicating that the viability of cell
lines decreased because of the downregulation of miR-
19a. A comparison with the negative control group, suggests that the miR19a suppression decreased the
proliferation of the cells.
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The Expression Level of miR-19a in Myeloma Cell
Lines was Reduced Upon BTZ Treatment
The expression level of miR-19a was evaluated after
BTZ treatment by qRT-PCR analysis, and our data
showed that its expression level was compared with
non-treated cell lines, reduced after treatment. In the
meantime, this expression was also evaluated in cells
transfected by the LentimiRa-off-has-mir-19a-3p vector
and the data showed that miR-19a expression level
was highly reduced after BTZ treatment, compared to
non-treated and non-transfected controls (Fig. 2c and
3c). Cell viability evaluation in 72 hours with Propidium
Iodide (PI) using flow cytometry showed that it is possible to evaluate every analysis on transfected cell
lines in 72 hours after transfection and on the other
hand, the viability of cell lines decreased due to the
downregulation of miR-19a (Fig. 2d and 3d).
Anti-miR-19a Increased Susceptibility to Bortezomib-
Induced Apoptosis
In order to investigate that antago-miR-19a affects
drug-induced apoptosis of myeloma cells, the percentage
of apoptotic cells by Annexin -V, PI through flow
cytometry after transfection of myeloma cell lines by
lentimiRa-off-has-mir-19a-3p and incubation with
bortezomib (BTZ, final concentration 5 ?M for U266
and 150 nM for RPMI8226) for 48 hours were analyzed.
Analysis of flow cytometry data showed that the
percentage of cells that underwent apoptosis increased
after transfection, compared with non-transfected cell lines and the negative control (Fig. 4a, b, c and d)
(RPMI8226: 23.5% Vs. 68.2 p=0.0038 U266: 25.2 vs.
96.4 p=0.0006). The data showed that antago-miR-19a
could increase myeloma cell susceptibility to drug-induced
apoptosis.
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SOCS3 and STAT3 mRNA expression level upregulated and downregulated, respectively after transfection of myeloma cell lines with LentimiRa-off-has-mir- 19a-3p vector.
After recognizing SOCS3 as the target of miR-19a, SOCS3 and its target STAT3 mRNA level in myeloma cell lines after transfection with Lentini-off-has-mir- 19a-3p vector was evaluated. The data showed that the mRNA expression level of SOCS3 increased, while STAT3 mRNA expression level decreased, compared with non-transfected cell lines as the negative control (Fig. 5a, b, 5c, and d).
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Discussion
Based on several findings of the studies previously conducted,[12] the first outcome of deregulated miRNA expression in MM, was reported by Loffler et al. in 2007,[13] which demonstrated miR-21 ectopic expression makes MM cells independent from the IL-6 growth stimulus. Pichiorri et al. identified a miRNA signature associated with the transformation of normal PCs to clinical MM via MGUS and demonstrated miR-32, miR-21, miR-17-92, miR-106b-25 cluster, and miR-181a/b were upregulated in both MM cell lines and primary tumors versus normal PCs: among them miR-17-92 cluster were highly expressed only in MM patients.[14] OncomiR-19a, as one of the members of miR-17-92 clusters has been proved to be involved in the promotion of cell proliferation, migration, and deduction of apoptosis, and suggests a critical role for it in the pathogenesis of myeloma.[10] Considering that better response in patients with the low level of miR-19a in serum and shortened progression- free with downregulation of it obtained from others studies,[15] we tried to evaluate the responsiveness of MM cell lines to BTZ in the presence of antagomiR- 19a to find a way to evade from resistance to it through targets of miR-19a. Recent studies figured out that SOCS3 is a target of miR-19a and indicated it is a negative regulator of SOCS3 [16,17] and showed that the molecule beneath it, i.e. STAT3, is a significant promoter of cancers such as MM when activated. [5,18,19] On the other hand STAT3, one of the components of the JAK/STAT pathway acts as an oncogene in human cancers.[19,20] Some miRNAs have close relativity with drug resistance, for example, past studies have shown that miR-181a expression is consistent with MM tumor load and could be a biomarker for treatment monitoring as much as miR-20a that showed the potential of a diagnostic biomarker.[4,6] BTZ as a proteasome inhibitor is an effective drug in the treatment of MM; however, drug resistance is still a major problem in 60% of patients.[8,21] So our data primarily showed that antagomir-19a down-regulated the miR-19a expression, and BTZ caused inhibition of miR-19a in a concentration-dependent manner and thereby in the presence of antago-miR-19a and BTZ, miR-19a efficiently downregulated in parallel with an increase in the proportion of apoptotic cells following treatment with BTZ. On the other hand, our viability studies showed that the ratio of the proliferation of myeloma cell lines decreased after transfection with antagomiR-19a vector. The data are consistent with other studies that proved miR-181a and miR-20a inhibited by BTZ.[6] In another part of our studies, we found out that downregulation of STAT3 and upregulation of SOCS3 at mRNA level which confirmed the reality of miR-19a function on SOCS3 is a negative regulator, the same as previous data.[16,17] As much as, the oncogenic function of STAT3 has been previously proved,[18,19] we could conclude that inhibition of miR-19a causes suppression of STAT3; and as indicated in our results, the expression level of STAT3 in BTZ treatment highly decreased using it in parallel with anatago-miR-19a. So, it can be argued that miR- 19a inhibition approach could be used to improve BTZ responsiveness in myeloma cells.The results are honorable & supplement the findings that microRNAs are differentially expressed in BTZ-resistant myeloma cells. So miR-19a could be proposed as a prognostic biomarker for responsiveness to BTZ in MM patients. To overcome the resistance and improve the level of responsiveness to BTZ, miR-19a targets such as SOCS3 and STAT3 could be tracked. We have faced some restrictions in our studies that must be adverted. They include the use of other MM cell lines and myeloma cells obtained from MM patients, and the evaluation of targets in protein level expression and downstream molecules, that could have helped us to generalize our results.
Conclusion
Overall, our data propose that induction of antago- miR-19a in myeloma cell lines resulted in a downregulation of miR-19a and enhancement of the level of responsiveness to BTZ treatment. On the other hand, the ratio of apoptosis in BTZ-treated cell lines was drastically more effective in the presence of miR-19a inhibition, so miR-19a could be suggested as a prognostic biomarker in MM treatment.Peer-review: Externally peer-reviewed.
References
1) Caracciolo D, Montesano M, Altomare E, Scionti F,
Di Martino MT, Tagliaferri P, et al. The potential role
of miRNAs in multiple myeloma therapy. Expert Rev
Hematol 2018;11(10):793-803.
2) Szymczyk A, Macheta A, Podhorecka M. Abnormal
microRNA expression in the course of hematological
malignancies. Cancer Manag Res 2018;10:4267-77.
3) Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA,
Jemal A. Global cancer statistics 2018: GLOBOCAN
estimates of incidence and mortality worldwide
for 36 cancers in 185 countries. CA Cancer J Clin
2018;68(6):394-424.
4) Zhang X, Chen Y, Zhao P, Zang L, Zhang Z, Wang X.
MicroRNA-19a functions as an oncogene by regulating
PTEN/AKT/pAKT pathway in myeloma. Leukemia
& lymphoma 2017;58(4):932-40.
5) Yin Z, Yang J, Ning R, Liu Y, Feng M, Gu C, et al. Signal
pathways, diseases, and functions associated with
the miR?19a/92a cluster and the use of berberine to
modulate the expression of this cluster in multiple myeloma
cells. J Biochem Mol Toxicol 2018;32(6):e22057
6) Peng J, Thakur A, Zhang S, Dong Y, Wang X, Yuan
R, et al. Expressions of miR-181a and miR-20a in
RPMI8226 cell line and their potential as biomarkers
for multiple myeloma. Tumor Biol 2015;36(11):8545-52.
7) Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath
S, Irwin D, et al. A phase 2 study of bortezomib
in relapsed, refractory myeloma. N Engl J Med
2003;348(26):2609-17.
8) Niewerth D, Jansen G, Assaraf YG, Zweegman S,
Kaspers GJ, Cloos J. Molecular basis of resistance to
proteasome inhibitors in hematological malignancies.
Drug Resist Updat 2015;18:18-35.
9) Lv H, Wu X, Ma G, Sun L, Meng J, Song X, et al.
An integrated bioinformatical analysis of miR-19a
target genes in multiple myeloma. Exp Ther Med
2017;14(5):4711-20.
10) Wang Y, Zhao S, Zhu L, Zhang Q, Ren Y. MiR-19a
negatively regulated the expression of PTEN and
promoted the growth of ovarian cancer cells. Gene
2018;670:166-73.
11) Zhu B, Ju S, Chu H, Shen X, Zhang Y, Luo X, et al. The
potential function of microRNAs as biomarkers and
therapeutic targets in multiple myeloma. Oncol Lett 2018;15(5):6094-106.
12) Peng Y, Croce CM. The role of MicroRNAs in human
cancer. Signal Transduct Target Ther. 2016 Jan
28;1:15004.
13) Loffler D, Brocke-Heidrich K, Pfeifer G, Stocsits C,
Hackermuller J, Kretzschmar A K, et al. Interleukin-
6 dependent survival of multiple myeloma cells
involves the STAT3-mediated induction of microRNA-
21 through a highly conserved enhancer. Blood
2007;110(4):1330-33.
14) Pichiorri F, Suh S S, Ladetto M, Kuehl M, Palumbo
T, Drandi D, et al. MicroRNAs regulate critical genes
associated with multiple myeloma pathogenesis. Proc
Natl Acad Sci USA 2008;105(35):12885-90.
15) Federico C, Sacco A, Belotti A, Ribolla R, Cancelli
V, Giacomini A, et al. Circulating microRNAs and
their role in multiple myeloma. Non-coding RNA
2019;5(2):37.
16) Collins AS, McCoy CE, Lloyd AT, O"Farrelly C, Stevenson
N J. miR-19a: an effective regulator of SOCS3
and enhancer of JAK-STAT signaling. PLoS One
2013;8(7):e69090.
17) Mahony R, Ahmed S, Diskin C, Stevenson N J. SOCS3
revisited: a broad regulator of disease, now ready for
therapeutic use? Cell Mol Life Sci 2016;73(17):3323-36.
18) He G, Karin M. NF-kappaB, and STAT3 - key players
in liver inflammation and cancer. Cell Res
2011;21(1):159-68.
19) Bromberg J. Stat proteins and oncogenesis. J Clin Invest
2002;109(9):1139-42.